Spinoculation Protocol

The MISSION® TRC shRNA libraries are lentiviral based shRNA vector collections for use in gene knockdown studies. This protocol describes the use of MISSION TRC shRNA Lentiviral Particles particularly for long term silencing and phenotypic observation in suspension cells. The following protocol has been developed from the literature1 for use in a 6-well plate with MISSION lentiviral particles.

The ExpressMag™ system (SHM01 or SHM02) has also been optimized to transduce suspension cells in 24 hours less time than this protocol and in multiple tissue culture formats.

Utilize SHC002V or SHC001V for optimizing transduction protocol. Transduction of SHC003V can be monitored via FACS or fluorescence microscopy, but protocols for this are outside of the scope of this document. It is critical to first optimize your protocol, so that you are confident in your transduction efficiency.This will deconvolute effects of the lentiviral payload (either shRNA or a custom payload) from transduction efficiency. Then, when switching to a MISSION lentivirus that expresses an shRNA, any changes in the resultant number of colonies or viable cells can be attributed to effects of the shRNA and cannot be attributable to a low transduction efficiency. After transduction efficiency has been optimized, utilize MISSION lentiviral particles expressing shRNA targeting your gene of interest or utilize a custom MISSION lentiviral particle expressing any shRNA or gene of interest.

Determine the final puromycin concentration to be used in the selection. The exact concentration is cell line dependent and should be determined by a 72 hour cytotoxicity assay on non-transduced cells.

Utilize only sterile items and work using standard tissue culture sterility techniques throughout this procedure.

Pre-warm all media to 37 °C.

Count cultured viable Jurkat cells.

Dilute Jurkat cells into complete medium to a final concentration of 1x105 cells/mL with a final volume of 14 mL.

Transfer 2 mL of 1x105 cells/mL into each of six sterile 15 mL conical tubes.

Use the equation below to calculate the MOI in a well. The ViralTiter will be provided in your certificate of analysis from Sigma. The VolumeofVirus is the volume in mL that is added to the well/centrifuge tube containing you the cells to be transduced. The NumberofCells is equal to the total number of viable cells in the well/centrifuge tube.